New parameters for in vitro development of cell lines of the species Astyanax bimaculatus (Linnaeus, 1758) and Geophagus proximus (Castelnau, 1855).

Intending to compare in vitro cell growth in different conditions, we established cell cultures using fin biopsies of two freshwater fishes, Astyanax bimaculatus and Geophagus proximus. Three different culture media (Leibovitz-L-15, Dulbecco's Modified Eagle Medium [DMEM], and 199) were employed, with or without the addition of AmnioMax, maintaining a standard temperature of 29°C. Based on the results obtained, we standardized a cell growth protocol in which medium 199 was less efficient for both species. Notably, G. proximus cells exhibited superior proliferation in DMEM and L-15 media, whereas A. bimaculatus cells demonstrated better parameters exclusively in the DMEM medium. Successful subculturing of cells with good proliferation index was observed, accompanied by preserved morphological characteristics. Therefore, the methodology outlined in this study represents an advancement in establishing fish cell cultures.

Canine transmissible venereal tumor - From general to molecular characteristics: A review.

Cancer is a group of complex diseases resulting from the accumulation of genetic and epigenetic changes affecting control and activity of several genes, especially those involved in cell differentiation and growth processes, leading to an abnormal proliferation. When the disease reaches an advanced stage, cancer can lead to metastasis in other organs. Interestingly, recent studies have shown that some types of cancer spread not only through the body, but also can be transmitted among individuals. Therefore, these cancers are known as transmissible tumors. Among the three types of transmissible tumors that occur in nature, the canine transmissible venereal tumor (CTVT) is known as the oldest cancer in the world, since it was originated from a single individual 11 000 years ago. The disease has a worldwide distribution, and its occurrence has been documented since 1810. The CTVT presents three types of cytomorphological classification: lymphocytoid type, mixed type, and plasmacytoid type, the latter being chemoresistant due to overexpression of the ABCB1 gene, and consequently increase of the P-glycoprotein. More knowledge about the epidemiology and evolution of CTVT may help to elucidate the pathway and form of the global spread of the disease.

Intracellular accumulation and DNA damage caused by methylmercury in glial cells.

Mercury is widely used in industrial and extractive processes, and the improper disposal of waste or products containing this metal produces a significant impact on ecosystems, causing adverse effects on living organisms, including humans. Exposure to methylmercury, a highly toxic organic compound, causes important neurological and developmental impairments. Recently, the genotoxicity of mercurial compounds has gained prominence as one of the possible mechanisms associated with the neurological effects of mercury, mostly by disturbing the mitotic spindle and causing chromosome loss. In this sense, it is important to investigate if these compounds can also cause direct damage to DNA, such as single and double-strand breaks. Thus, the aim of this study was to investigate the cytotoxic and genotoxic potential of methylmercury in cell lines derived from neurons (B103) and glia (C6), exposed to methylmercury (MeHg) for 24 h, by analyzing cell viability, metabolic activity, and damage to DNA and chromosomes. We found that in comparison to the neuronal cell line, glial cells showed higher tolerance to MeHg, and therefore a higher LC50 and consequent higher intracellular accumulation of Hg, which led to the occurrence of several genotoxic effects, as evidenced by the presence of micronuclei, bridges, sprouts, and chromosomal aberrations.

Comparative Cytogenetics in Four Leptodactylus Species (Amphibia, Anura, Leptodactylidae): Evidence of Inner Chromosomal Diversification in Highly Conserved Karyotypes.

With 82 species currently described, the genus Leptodactylus is the most diverse and representative one in the family Leptodactylidae. Concerning chromosomal organization, this genus represents an interesting and underexplored group since data from molecular cytogenetics are incipient, and little is known about the organization and distribution of repetitive DNA elements in the karyotypes. In this sense, this study aimed at providing a comparative analysis in 4 Leptodactylus species (L. macrosternum, L. pentadactylus, L. fuscus, and Leptodactylus cf. podicipinus), combining conventional cytogenetics (Giemsa staining, C-banding, and AgNOR staining) and mapping of molecular markers (18S rDNA, telomeric and microsatellite probes), to investigate mechanisms underlying their karyotype differentiation process. The results showed that all species had karyotypes with 2n = 22 and FN = 44, except for Leptodactylus cf. podicipinus which presented FN = 36. The 18S rDNA was observed in pair 8 of all analyzed species (corresponding to pair 4 in L. pentadactylus), coinciding with the secondary constrictions and AgNOR staining. FISH with microsatellite DNA probes demonstrated species-specific patterns, as well as an association of these repetitive sequences with constitutive heterochromatin blocks and ribosomal DNA clusters, revealing the dynamics of microsatellites in the genome of the analyzed species. In summary, our data demonstrate an ongoing process of genomic divergence inside species with almost similar karyotype, driven most likely by a series of pericentric inversions, followed by differential accumulation of repetitive sequences.

The Karyotype of Salvator merianae (Squamata, Teiidae): Analyses by Classical and Molecular Cytogenetic Techniques.

In this study, we analyzed the karyotype of Salvator merianae (Teiidae) from the Brazilian semiarid region using different cytogenetic markers. Chromosomes were examined by classical (Giemsa and AgNOR staining) and molecular (FISH with ribosomal, telomeric, and microsatellite probes) cytogenetic approaches. S. merianae showed a diploid chromosome number of 2n = 38 (10 biarmed macrochromosomes + 28 microchromosomes). No sex-linked chromosome heteromorphisms were observed. Clusters of 18S/28S rDNA were localized in the terminal region of the long arm of pair 2. In addition to the typical telomeric signals, (TTAGGG)n repeats were detected in the pericentromeric region of some macrochromosome pairs, which might indicate the occurrence of chromosomal rearrangements via chromosome fusions. Hybridization signals of the microsatellite probes (GA)n, (GAA)n, and (GAG)n were uniformly distributed across all chromosomes, while (CA)n, (CAA)n, and (CAC)n produced brighter signals in the telomeric and pericentromeric regions of specific chromosome pairs. The comparison with previous studies demonstrates that, despite the wide distribution of S. merianae, the macrostructure organization of the karyotype remained unchanged, showing stability in diploid number and chromosome morphology.

Evolution of Bird Sex Chromosomes Narrated by Repetitive Sequences: Unusual W Chromosome Enlargement in Gallinula melanops (Aves: Gruiformes: Rallidae).

Among birds, species with the ZZ/ZW sex determination system generally show significant differences in morphology and size between the Z and W chromosomes (with the W usually being smaller than the Z). In the present study, we report for the first time the karyotype of the spot-flanked gallinule (Gallinula melanops) by means of classical and molecular cytogenetics. The spot-flanked gallinule has 2n = 80 (11 pairs of macrochromosomes and 29 pairs of microchromosomes) with an unusual W chromosome that is larger than the Z. Besides being totally heterochromatic, it has a secondary constriction in its long arm corresponding to the nucleolar organizer region, as confirmed by both silver staining and mapping of 18S rDNA probes. This is an unprecedented fact among birds. Additionally, 18S rDNA sites were also observed in 6 microchromosomes, while 5S rDNA was found in just 1 microchromosomal pair. Seven out of the 11 used microsatellite sequences were found to be accumulated in microchromosomes, and 6 microsatellite sequences were found in the W chromosome. In addition to the involvement of heterochromatin and repetitive DNAs in the differentiation of the large W chromosome, the results also show an alternative scenario that highlights the plasticity that shapes the evolutionary history of bird sex chromosomes.

Karyotype Evolution and Distinct Evolutionary History of the W Chromosomes in Swallows (Aves, Passeriformes).

As in many other bird groups, data on karyotype organization and distribution of repetitive sequences are also lacking in species belonging to the family Hirundinidae. Thus, in the present study, we analyzed the karyotypes of 3 swallow species (Progne tapera, Progne chalybea, and Pygochelidon cyanoleuca) by Giemsa and AgNOR staining, C-banding, and FISH with 11 microsatellite sequences. The diploid chromosome number was 2n = 76 in all 3 species, and NORs were observed in 2 chromosome pairs each. The microsatellite distribution pattern was similar in both Progne species, whereas P. cyanoleuca presented a distinct organization. These repetitive DNA sequences were found in the centromeric, pericentromeric, and telomeric regions of the macrochromosomes, as well as in 2 interstitial blocks in the W chromosome. Most microchromosomes had mainly telomeric signals. The Z chromosome displayed 1 hybridization signal in P. tapera but none in the other species. In contrast, the W chromosome showed an accumulation of different microsatellite sequences. The swallow W chromosome is larger than that of most Passeriformes. The observed enlargement in chromosome size might be explained by these high amounts of repetitive sequences. In sum, our data highlight the significant role that microsatellite sequences may play in sex chromosome differentiation.

Comparative Chromosome Painting in Two Brazilian Stork Species with Different Diploid Numbers.

Despite the variation observed in the diploid chromosome number of storks (Ciconiiformes, Ciconiidae), from 2n = 52 to 2n = 78, most reports have relied solely on analyses by conventional staining. As most species have similar macrochromosomes, some authors propose that karyotype evolution involves mainly fusions between microchromosomes, which are highly variable in species with different diploid numbers. In order to verify this hypothesis, in this study, the karyotypes of 2 species of storks from South America with different diploid numbers, the jabiru (Jabiru mycteria, 2n = 56) and the maguary stork (Ciconia maguary, 2n = 72), were analyzed by chromosome painting using whole chromosome probes from the macrochromosomes of Gallus gallus (GGA) and Leucopternis albicollis (LAL). The results revealed that J. mycteria and C. maguary share synteny within chromosome pairs 1-9 and Z. The syntenies to the macrochromosomes of G. gallus are conserved, except for GGA4, which is homologous to 2 different pairs, as in most species of birds. A fusion of GGA8 and GGA9 was observed in both species. Additionally, chromosomes corresponding to GGA4p and GGA6 are fused to other segments that did not hybridize to any of the macrochromosome probes used, suggesting that these segments correspond to microchromosomes. Hence, our data corroborate the proposed hypothesis that karyotype evolution is based on fusions involving microchromosomes. In view of the morphological constancy of the macrochromosome pairs in most Ciconiidae, we propose a putative ancestral karyotype for the family, including the GGA8/GGA9 fusion, and a diploid number of 2n = 78. The use of probes for microchromosome pairs should be the next step in identifying other synapomorphies that may help to clarify the phylogeny of this family.

The Karyotype of the Hoatzin (Opisthocomus hoazin) - A Phylogenetic Enigma of the Neornithes.

The hoatzin (Opisthocomus hoazin Müller, 1776) is a folivorous bird, endemic to the Amazonian region. It presents some unique characteristics, including wing claws and foregut fermentation, which make its phylogenetic relationship to other birds difficult to determine. There have been various attempts to place it among the Galliformes, Gruiformes, Musophagiformes, Cuculiformes, and Charadriiformes, but phylogenetic analyses always show low supporting values. Nowadays, the hoatzin is included in the monotypic order Opisthocomiformes, but the relationship of this order to other groups of birds is still unclear. Although its karyotype resembles the typical avian model, fissions of the syntenic groups corresponding to chicken chromosomes 1 and 2 and 2 fusions were found. The presence of 18S rDNA clusters in 2 pairs of microchromosomes is another derived character. Hence, different rearrangements were detected in the karyotype of the hoatzin, indicating it has been derived from the putative ancestral karyotype by the occurrence of fissions and fusions. However, as these rearrangements are not exclusive to O. hoazin, they do not clarify the phylogenetic position of this enigmatic species.

Chromosome Painting in Trogon s. surrucura (Aves, Trogoniformes) Reveals a Karyotype Derived by Chromosomal Fissions, Fusions, and Inversions.

Trogons are forest birds with a wide distribution, being found in Africa, Asia, and America, and are included in the order Trogoniformes, family Trogonidae. Phylogenetic studies using molecular data have not been able to determine the phylogenetic relationship among the different genera of trogons. So far, no cytogenetic data for these birds exist. Hence, the aim of this study was to characterize the karyotype of Trogon surrucura surrucura by means of classical and molecular cytogenetics. We found a diploid chromosome number of 2n = 82, similar to most birds, with several derived features compared to chicken and the putative ancestral avian karyotype. T. s. surrucura showed 3 pairs of microchromosomes bearing 18S rDNA clusters. The Z and W sex chromosomes were of similar size but could readily be identified by morphological differences. Using chromosome painting with whole chromosome probes from Gallus gallus and Leucopternis albicollis, we found that the chromosomes homologous to chicken chromosomes 2 and 5 correspond to 2 different pairs in T. s. surrucura and L. albicollis, due to the occurrence of centric fissions. Paracentric inversions were detected in the segment homologous to chicken chromosome 1q, and we confirmed the recurrence of breakpoints when our results were compared to other species of birds already analyzed by FISH or by in silico genome assembly.

Chromosome Painting in Tyrant Flycatchers Confirms a Set of Inversions Shared by Oscines and Suboscines (Aves, Passeriformes).

Tyrannidae is the largest family of Passeriformes in the Neotropical region. However, despite an interesting chromosomal diversity, there are only few cytogenetic studies of this family, and most of these are based on conventional cytogenetics. Hence, we analyzed here the chromosomal diversity and karyotypical evolution of this group by chromosome painting in 3 different species - Pitangus sulphuratus, Serpophaga subcristata, and Satrapa icterophrys - and make comparisons with previous data. In addition to chromosome painting with Gallus gallus (GGA) and Leucopternis albicollis (LAL) probes, karyotypes were analyzed by conventional staining, C-banding, and FISH with 18S rDNA and telomeric probes. Although this family is characterized by extensive chromosomal variation, we found similar karyotypes and diploid numbers ranging from 2n = 80 in P. sulphuratus to 2n = 82 in S. subcristata and S. icterophrys. Constitutive heterochromatin was located centromerically in all 3 species. Clusters of 18S rDNA were present in 1 pair of microchromosomes, except in S. subcristata, where 2 pairs of microchromosomes were labeled. No interstitial telomeric sequences were detected. GGA and LAL whole-chromosome probes revealed the occurrence of fissions and both paracentric and pericentric inversions commonly seen in other Passeriformes. In general terms, tyrants show the typical karyotype found in Passeriformes, suggesting that the observed rearrangements occurred before the division of the suborders Oscines and Suboscines.

Chromosomal Mapping of Repetitive DNAs in Myiopsitta monachus and Amazona aestiva (Psittaciformes, Psittacidae) with Emphasis on the Sex Chromosomes.

Here, for the first time, we describe the karyotype of Myiopsitta monachus (Psittacidae, Arini). We found 2n = 48, corresponding to the lowest diploid number observed in Neotropical Psittaciformes so far, with an uncommonly large W chromosome homomorphic to the Z. In order to better understand the evolution of the sex chromosomes in this species, we applied several molecular cytogenetic approaches, including C-banding, FISH mapping of repetitive DNAs (several microsatellite repeats), and whole-chromosome painting on metaphases of M. monachus. For comparison, another species belonging to the same tribe but with a smaller W chromosome (A. aestiva) was also analyzed. The results show that the constitutive heterochromatin has a very diverse distribution pattern in these species revealing heterochromatic blocks in the centromeric region of all chromosomes and in most of the length of the W chromosome in A. aestiva, while in M. monachus they were found in interstitial and telomeric regions. Concerning the microsatellites, only the sequence (CG)n produced signals on the W chromosome of A. aestiva, in the distal region of both arms. However, in M. monachus, (CAA)n, (CAG)n, and (CG)n probes were accumulated on the W chromosome, and, in addition, the sequence (CAG)n also hybridized to heterochromatic regions in macrochromosomes, as well as in microchromosomes. Based on these results, we suggest that the increase in length of the W chromosome in M. monachus is due to the amplification of repetitive elements, which highlights their significant role in the evolutionary process of sex chromosome differentiation.

First Comprehensive Characterization of Phayre's Leaf-Monkey (Trachypithecus phayrei) Karyotype.

The chromosomal homologies of human (Homo sapiens-HSA) and Trachypithecus phayrei (TPH-Phayre's leaf-monkey, family Cercopithecidae) have previously been studied by using classical chromosome staining/banding and fluorescence in situ hybridization (FISH) from the 1970s to 1990s. In this study, we carried out molecular cytogenetics applying human multicolor banding (MCB), locus-specific, and human heterochromatin-specific probes to establish the first detailed chromosomal map of TPH, which was not available until now. Accordingly, it was possible to precisely determine evolutionary-conserved breakpoints (ECBs) and the orientation of evolutionary-conserved segments compared to HSA. It could be shown that five chromosomes remained completely unchanged between these two species, and 16 chromosomes underwent only intrachromosomal changes. In addition, 50 ECBs that failed to be resolved in previous reports were exactly identified and characterized in this study. It could also be shown that 43.5% of TPH centromere positions were conserved and 56.5% were altered compared to HSA. Interestingly, 82% ECBs in TPH corresponded to human fragile sites. Overall, this study is an essential contribution to future studies and reviews on chromosomal evolution in Cercopithecidae.

Genomic Organization of Microsatellites and LINE-1-like Retrotransposons: Evolutionary Implications for Ctenomys minutus (Rodentia: Ctenomyidae) Cytotypes.

The Neotropical underground rodents of the genus Ctenomys (Rodentia: Ctenomyidae) comprise about 65 species, which harbor the most significant chromosomal variation among mammals (2n = 10 to 2n = 70). Among them, C. minutus stands out with 45 different cytotypes already identified, among which, seven parental ones, named A to G, are parapatrically distributed in the coastal plains of Southern Brazil. Looking for possible causes that led to such extensive karyotype diversification, we performed chromosomal mapping of different repetitive DNAs, including microsatellites and long interspersed element-1 (LINE-1) retrotransposons in the seven parental cytotypes. Although microsatellites were found mainly in the centromeric and telomeric regions of the chromosomes, different patterns occur for each cytotype, thus revealing specific features. Likewise, the LINE-1-like retrotransposons also showed a differential distribution for each cytotype, which may be linked to stochastic loss of LINE-1 in some populations. Here, microsatellite motifs (A)30, (C)30, (CA)15, (CAC)10, (CAG)10, (CGG)10, (GA)15, and (GAG)10 could be mapped to fusion of chromosomes 20/17, fission and inversion in the short arm of chromosome 2, fusion of chromosomes 23/19, and different combinations of centric and tandem fusions of chromosomes 22/24/16. These data provide evidence for a correlation between repetitive genomic content and localization of evolutionary breakpoints and highlight their direct impact in promoting chromosomal rearrangements.

Karyotypical Confirmation of Natural Hybridization between Two Manatee Species, Trichechus manatus and Trichechus inunguis.

Two species of manatees are found in Northern Brazil-the Antillean manatee (Trichechus manatus), which is found along the coast from Florida to Northeastern Brazil, and the Amazonian manatee (Trichechus inunguis), endemic to the Amazon drainage basin. These species show a sympatric distribution in the region of the Marajó Archipelago, an estuarine area surrounding the Amazon River mouth. There is evidence of the occurrence of interspecific hybrids in this area, based on mitochondrial DNA analyses, although the use of nuclear markers has not corroborated this proposal. Considering that these species show very distinct karyotypes, despite being closely related (2n = 48 in T. manatus and 2n = 56 in T. inunguis), hybrids would present distinct chromosome numbers. Based on this, we conducted cytogenetic analyses using classic and molecular techniques in three calves found stranded in the Marajó Island and Amapá coast. The results showed that one of them, morphologically classified as T. inunguis, presented the correspondent karyotype, with 2n = 56. However, the other two, which were phenotypically similar to T. manatus, showed 2n = 49. Despite the same diploid number, their G-banding patterns revealed some differences. The results of the distribution of some microsatellite sequences have also confirmed the heterozygosity of some chromosomal pairs in these two individuals. These results are the first indubitable confirmation of the occurrence of natural hybrids between T. manatus and T. inunguis, and also brings about some issues concerning the viability of hybrids, considering that these two individuals do not correspond to an F1 hybrid, but instead, both presented a possible F2 karyotype.

Recurrent chromosome reshuffling and the evolution of neo-sex chromosomes in parrots.

The karyotype of most birds has remained considerably stable during more than 100 million years' evolution, except for some groups, such as parrots. The evolutionary processes and underlying genetic mechanism of chromosomal rearrangements in parrots, however, are poorly understood. Here, using chromosome-level assemblies of four parrot genomes, we uncover frequent chromosome fusions and fissions, with most of them occurring independently among lineages. The increased activities of chromosomal rearrangements in parrots are likely associated with parrot-specific loss of two genes, ALC1 and PARP3, that have known functions in the repair of double-strand breaks and maintenance of genome stability. We further find that the fusion of the ZW sex chromosomes and chromosome 11 has created a pair of neo-sex chromosomes in the ancestor of parrots, and the chromosome 25 has been further added to the sex chromosomes in monk parakeet. Together, the combination of our genomic and cytogenetic analyses characterizes the complex evolutionary history of chromosomal rearrangements and sex chromosomes in parrots.

Maintenance of syntenic groups between Cathartidae and Gallus gallus indicates symplesiomorphic karyotypes in new world vultures.

Similarities between New World and Old World vultures have been interpreted to reflect a close relationship and to suggest the inclusion of both in Accipitridae (Falconiformes). However, deeper analyses indicated that the placement of the New World vultures (cathartids) in this Order is uncertain. Chromosome analysis has shown that cathartids retained a karyotype similar to the putative avian ancestor. In order to verify the occurrence of intrachromosomal rearrangements in cathartids, we hybridized whole chromosome probes of two species (Gallus gallus and Leucopternis albicollis) onto metaphases of Cathartes aura. The results showed that not only were the syntenic groups conserved between Gallus and C. aura, but probably also the general gene order, suggesting that New World vultures share chromosomal symplesiomorphies with most bird lineages.

Metabolic and oxidative impairments in human salivary gland cells line exposed to MeHg.

BACKGROUND/AIM: The ingestion of contaminated seafood by MeHg is considered the main route of human exposure, turning the salivary gland one important target organ. The salivary glands play critical roles in maintaining oral health homeostasis, producing saliva that maintains the oral microbiota, initiation of the digestion of macromolecules, and being essential in maintaining the integrity of the adjacent soft tissues and teeth. Thus, this study aimed to investigate the effects of MeHg exposure on human salivary gland cells line. METHODS: Cells were exposed to 1-6 µM of MeHg for 24 h, and analysis of toxicity was performed. Based on these results, the LC50 was calculated and two concentrations were chosen (0.25 and 2.5 µM MeHg) to evaluate intracellular mercury (Hg) accumulation (THg), metabolic viability and oxidative stress parameters (GSH:GSSG ratio, lipid peroxidation, protein oxidation and DNA damage). RESULTS: The results demonstrated accumulation of THg as we increased the MeHg concentrations in the exposure and, the higher the dose, the lower is the cell metabolic response. In addition, the 2.5 µM MeHg concentration also triggered oxidative stress in human salivary gland cells by depleting the antioxidant competence of GSH:GSSG ratio and increasing lipid peroxidation and proteins carbonyl levels, but no damages to DNA integrity. CONCLUSION: In conclusion, although these two elected doses did not show lethal effects, the highest dose triggered oxidative stress and new questionings about long-term exposure models are raised to investigate furthers cellular damages to human salivary gland cells caused by MeHg exposure to extrapolate in a translational perspective.

Non-Lethal Concentration of MeHg Causes Marked Responses in the DNA Repair, Integrity, and Replication Pathways in the Exposed Human Salivary Gland Cell Line.

In Brazilian northern Amazon, communities are potentially exposed and vulnerable to methylmercury (MeHg) toxicity through the vast ingestion of fish. In vivo and in vitro studies demonstrated that the salivary glands as a susceptible organ to this potent environmental pollutant, reporting alterations on physiological, biochemical, and proteomic parameters. However, the alterations caused by MeHg on the gene expression of the exposed human salivary gland cells are still unknown. Therefore, the goal was to perform the transcriptome profile of the human salivary gland cell line after exposure to MeHg, using the microarray technique and posterior bioinformatics analysis. The cell exposure was performed using 2.5 µM MeHg. A previously published study demonstrated that this concentration belongs to a range of concentrations that caused biochemical and metabolic alterations in this linage. As a result, the MeHg exposure did not cause lethality in the human salivary gland cells line but was able to alter the expression of 155 genes. Downregulated genes (15) are entirety relating to the cell metabolism impairment, and according to KEGG analysis, they belong to the glycosphingolipid (GSL) biosynthesis pathway. On the other hand, most of the 140 upregulated genes were related to cell-cycle progression, DNA repair, and replication pathway, or cellular defenses through the GSH basal metabolism. These genomic changes revealed the effort to the cell to maintain physiological and genomic stability to avoid cell death, being in accordance with the nonlethality in the toxicity test. Last, the results support in-depth studies on nonlethal MeHg concentrations for biomarkers identification that interpret transcriptomics data in toxicological tests serving as an early alert of physiological changes in vitro biological models.

Gene Expression Profile in Immortalized Human Periodontal Ligament Fibroblasts Through hTERT Ectopic Expression: Transcriptome and Bioinformatic Analysis.

Human periodontal ligament fibroblast (hPLF) cells play an important role in maintaining oral cavity homeostasis with special function in tissue regeneration and maintenance of dental alveoli. Although their primary cell cultures are considered a good experimental model with no genetic changes, the finite life span may limit some experimental designs. The immortalization process increases cell life span but may cause genetic changes and chromosomal instability, resulting in direct effects on physiological cell responses. In this way, we aimed to investigate the global gene expression of hPLFs after the immortalization process by the ectopic expression of the catalytic subunit of the enzyme telomerase reverse transcriptase (hTERT) through transcriptome analysis. The embryonic origin of the primary culture of hPLF cells and immortalized hPLF-hTERT was also tested by vimentin staining, hTERT synthesis evaluated by indirect immunocytochemistry, analysis of cell proliferation, and morphology. The results indicated that hPLFs and hPLF-hTERT were positive for vimentin. On the 20th cell passage, hPLFs were in senescence, while hPLF-hTERT maintained their proliferation and morphology characteristics. At the same passage, hPLF-hTERT presented a significant increase in hTERT synthesis, but transcriptome did not reveal overexpression of the hTERT gene. Fifty-eight genes had their expression altered (11 upregulated and 47 downregulated) with the absence of changes in the key genes related to these cell types and in the main cancer-associated genes. In addition, the increase in hTERT protein expression without the overexpression of its gene indicates posttranscriptional level regulation. Successful immortalization of hPLFs through the ectopic expression of hTERT encourages further studies to design experimental protocols to investigate clinical questions from a translational perspective.